In recent years, in fields of medicine production, gene therapy, regenerative medicine, immunotherapy and the like, it is required that cells, tissues, microorganisms or the like are efficiently cultured in large amounts under an artificial environment, and the cells are automatically cultured in large amounts by use of a culture bag having a gas permeability in a closed system.
Additionally, in recent years, there have rapidly been developed research and development of the regenerative medicine and the like in which iPS cells having a versatile differentiation potency, mesenchymal stem cells having a differentiation potency to cells belonging to mesenchymal series, or the like are particularly used. Most of the cells for use in such research and development are adhesive culture cells.
The adhesive culture cells are cells that proliferate adhering to a culture container, and hence, these cells are different from floating cells that proliferate in a floating state in a culture liquid in that it is necessary to release the cultured cells from the culture container. Consequently, the adhesive culture cells have heretofore been often cultured in an opened culture container such as a petri dish to enable a releasing operation.
However, culturing the cells in large amounts in the petri dish is complicated and requires a great deal of time and labor, and hence, to efficiently culture the cells in large amounts in future, it is desirable to carry out the cultivation by use of a closed culture bag.
Additionally, in culturing the adhesive culture cells, it is necessary to only culture the selected cells having an excellent condition or the like, and hence it is also required that a desirable portion is only released from the culture container.
As a conventional cell release method, a trypsin treatment can first be quoted. In this method, an adhesion factor of the adhesive culture cells to the culture container is decomposed with trypsin that is a proteolytic enzyme, whereby the cells are released.
That is, as shown in FIG. 8, a petri dish 10 into which a culture liquid 2 is placed is seeded with adhesive culture cells 3 to proliferate the cells, and then the culture liquid 2 is removed from the petri dish 10 shown in step (1) of FIG. 8. Next, culture liquid components are washed with phosphate buffered saline (a PBS solution) shown in step (2) of FIG. 8, and a trypsin solution is poured thereinto shown in step (3) of FIG. 8. The solution is held in an incubator for several minutes to decompose the adhesion factor, and the adhesive culture cells 3 are released in pieces from the petri dish 10 shown in step (4) of FIG. 8. Finally, the culture liquid 2 is poured into the petri dish 10 to devitalize trypsin, and the adhesive culture cells 3 are collected together with the culture liquid 2 shown in step (5) of FIG. 8.
According to such a trypsin treatment, the adhesive culture cells 3 can be released from the culture container.
In addition, as a conventional cell release method, there is also a method which physically scrapes cells by use of a cell scraper (see Patent Document 1). According to this method, an operation is easier than in the trypsin treatment, and cells in a targeted range can be released to a certain degree.
Furthermore, Patent Document 2 discloses a method in which a scanning type probe microscope having a probe is utilized and the probe is pressed onto specific cells with a predetermined force to apply a physical stimulus to the cells, thereby releasing the cells from a substrate. According to this method, the cells can selectively be released.
Patent Document 1: Japanese Utility Model Registration No. 2531969
Patent Document 2: Japanese Patent No. 4831543